Structural and functional nuclear organization of the beta-globin gene locus in vivo.

The DNA sequence of the human genome has provided a tremendous insight into our genetic make-up. However the DNA sequence alone has provided few clues as to how genetic information is controlled so that the correct genes are activated at the right time during development and in the proper tissues. We have found that gene regulation is extremely complex with multiple levels of control. Cells package inactive genomic regions into compact structures within the nucleus that may play a role in keeping the genes in those regions silent. Active genes on the other hand are relatively decondensed and highly mobile within the confines of a defined nuclear space. We have shown that individual genes communicate with distal genomic sequence that contain important regulatory information by direct physical contact. Futhermore we have shown that active genes often congregate in discrete nuclear locations that seem to be important for their expression. We will investigate the events necessary for the communication between genes and distal regulatory sequences and the congregation or organization of active genes in the nucleus. This information is vital to understand normal gene function as well as what goes wrong in cases were genes are deregulated such as in cancer.

The proposed experiments will investigate the higher-order structure (chromosome folding) and functional nuclear organization (placement in the nucleus) of genes during transcriptional activity and inactivity. We have found that widely separated genes on the same chromosome often co-localize when they are transcriptionally active, at frequencies much higher than would be expected by random association. Taken together with published reports, which show high chromatin mobility, individual genes adopting distinct nuclear localizations in association with transcriptional activity and nuclear sub-compartments rich in transcriptional apparatus, these data suggest that gene activity is controlled in part by nuclear organization. We have shown that the beta-globin genes and the distal beta-globin locus control region physically associate in vivo during the process of transcription. This higher-order structure appears to be dependent on modifications to the histone tails of chromatin in sub-domains encompassing the developmentally specific genes. These modifications in turn are dependent on intergenic transcription through the subdomain region suggesting that the process of transcription or the presence of the non-coding RNA targets remodelling and histone modification factors to the sub-domains. However another non-exclusive hypothesis is that intergenic transcription itself is involved in the assembly of higher-order structures by acting as a molecular motor to pull distal regions into functional complexes. We will investigate these models and attempt to determine a hierarchy of events that take place in the normal control of gene expression. We will use state-of-the-art techniques such as RNA TRAP, 3C, and 3D confocal RNA and DNA FISH to assess higher-order chromatin structures as well as coordinate nuclear organization of genes as far apart as 40 Mb. In addition we will develop a high-throughput assay to rapidly build up information about the functional organization of the nucleus from numerous genomic perspectives. This information is vital to understanding normal gene function as well as in cases were genes are deregulated as in cancer.